The dominant theory of aging is the mitochondrial free radical theory, which proposes that the production of reactive oxygen species (ROS) by intracellular mitochondria is the primary cause of cellular damage and aging. However, the theory is at best incomplete. To test it critically and to manipulate radical production to promote healthspan, it is therefore crucial to fully understand the mechanisms and specific sites of mitochondrial radical generation to assess its role in age-related diseases and aging, ultimately to allow rational design of beneficial therapies. Despite much research, such understanding is currently lacking. This study will address this deficit by measuring the rate of cellular mitochondrial ROS generation at each specific site in the electron transport chain in cells relevant to aging, particularly neurons, muscle cells and fibroblasts. Previous attempts to identify the sites involved have led to confusion because of a conceptual problem: to define the sites, electron transport inhibitors are added, but they inevitably distort electron distribution in the chain and the pattern of ROS production. This problem will be circumvented by a key innovation: using inhibitors to define sites in a first phase in which endogenous reporter signals are calibrated in terms of ROS production from each site, but omitting them in the measuring phase when the endogenous reporter signals are used to calculate ROS generation from each site under a single experimental condition when several sites may operate simultaneously. The hypothesis to be tested is that at least one site in the mitochondrial electron transport chain produces significant ROS in cells and is the main oxidative stress generator. In the mitochondrial free radical theory, ROS generation at this site would be the primary cause of aging and its diseases. The hypothesis will be investigated by a rigorous stepwise approach: first assay rates from each site in a model system (isolated mitochondria), then build on this platform to assay the sites of ROS production in cells. Studies will have the following specific aims. (1) Establish and validate measurements of ROS production from different sites in the electron transport chain of isolated mitochondria in the absence of electron transport inhibitors. (2) Using these methods, determine the relative rate of ROS production by each site in isolated mitochondria from different ageing-relevant tissues, oxidizing different substrates in different states, at different ages. (3) Transfer the methods to cells and establish the relative rate of ROS production by each mitochondrial site and the quantitative importance of mitochondrial ROS production. We will use synaptosomes and experimentally-amenable cells (including neurons, astrocytes, normal and senescent fibroblasts, C2C12 myoblasts, and pancreatic insulinoma cells oxidizing physiological substrates in different states. PUBLIC HEALTH RELEVANCE: Since age is the primary risk factor for many diseases, understanding the mechanisms of aging may allow us to significantly reduce the burden of disease and increase human healthspan - even a small decrease in age-related cellular damage may have widespread public health benefits. Free radicals generated during normal oxidative metabolism damage cells and may cause aging. This project will use an innovative stepwise approach to identify measure and compare the specific cellular sites at which these radicals are produced, opening the way for rational strategies to lower radical production and decrease the diseases of aging.